A nanopore DNA sequencer provided with a pore of a nanometer size (nanopore) which is almost equal to the size of DNA, and a sensor to read base sequences in the neighborhood or the inner portion thereof has paid attention as a method for determining base sequences of deoxyribonucleic acid (DNA) without using a reagent (NPL 1).
The nanopore DNA sequencer sequentially identifies base species by directly measuring changes in a physical quantity based on each base species contained in a DNA strand when the DNA strand passes through the nanopore. The sequencer is capable of high speed decoding, further does not amplify template DNA by an enzyme and does not use labeling substances such as fluorescent substances, and is thus expected to lead to high throughput, low costs and decoding of long base length. As changes in physical quantity based on base species, proposed are changes in tunnel current through a DNA strand, the charge amount of the DNA strand, and ion current passing through a nanopore and the like when DNA passes through the nanopore, and a method for measuring these and the like.
In nanopore DNA sequencing, a nanopore plays a role of controlling the transport of a single molecule of DNA strand. There are two major problems as follows. The first problem is to provide a method for stably producing a nanopore the size of which allows the passage of only a single molecule of DNA strand on a large scale. The second problem is to delay the velocity of a DNA strand passing through a nanopore to the velocity enough to read the base sequence of DNA. As a method for solving the above-mentioned problems, the following two approaches have been proposed.
The first approach is a method using a micropore formed by modified protein as a nanopore (bionanopore). It has been reported that a protein having a fine pore through which a single molecule of DNA strand selectively passes is produced and allowed to support the inside of membrane protein, thereby being able to control the transport of DNA (NPL 2). The second approach is a method in which a nanopore is produced in a solid thin film by a top-down means using a semiconductor micromachining process (solid-state nanopore). As one of typical production methods, there is a method in which a region containing a thin film insulator is provided on a semiconductor substrate and an electron beam is irradiated to form a pore as described in NPL 3. A fine pore with 10 nm or less can be formed by controlling the energy, area irradiated and current of the electron beam.
In the meantime, as a method for forming a very fine pattern within the process limitation of photolithography or less, a block copolymer lithography method using microphase separation, which is the self-assembly process of a block copolymer, has paid attention as a next-generation semiconductor lithography technique (NPL 4). Cylindrical and linear/spatial microdomains are formed by self-assembly by microphase separation of poly(styrene-b-methylmethacrylate) (PS-b-PMMA), a type of block copolymer, and a pattern with about 10 nm to 100 nm can be obtained by removing domains containing polymethylmethacrylate by etching. A method for processing a substrate using the microdomains as a mask has been considered.
Further, when a block copolymer containing a hydrophobic polymer chain and a hydrophilic polymer chain is used and fine cylinders containing the hydrophilic polymer chain are formed by self-assembly to penetrate a block copolymer thin film, it is shown that a dye molecule permeates the fine cylinders (NPL 5).